The role of TRIP-Br proteins in the regulation of mammalian gene transcription and cell cycle progression

Abstract

The TRIP-Br proteins (TRIP-Br1 and TRIP-Br2) are a novel family of transcriptional regulators that have been proposed to function as a??integratorsa?? at E2F-responsive promoters to integrate signals provided by PHD zinc finger- and/or bromodomain-containing transcription factors. Two approaches were employed o further probe the physiological function(s) of the TRIP-Br proteins, namely the decoy peptide strategy and the DNA enzyme strategy. Synthetic decoy peptides shown to antagonize the in vitro and in vivo interaction between TRIP-Br1 (*Br1) or TRIP-Br2 (*Br2) and the PHD zinc finger and/or bromodomain of other transcription factors, were introduced into U2OS cells to further elucidate the TRIP-Br integrator function(s). Both the *Br1 and *Br2 peptides were found to differentially down-regulate the expression of endogenous E2F-responsive genes in vivo, and impose a state of global cell cycle arrest. Thus, the integrator function of TRIP-Br proteins is required for proper execution of E2F-dependent mammalian cell cycle progression. Further analyses on synchronously cycling U2OS cells showed that treatment with *Br1 or *Br2 caused deregulated cyclin E protein accumulation during cell cycle progression. This was shown to be associated with the down-regulation of the Fbxw7 gene, a novel E2F-responsive and TRIP-Br co-regulated gene that encodes an ubiquitin ligase (E3) responsible for targeting cyclin E for ubiquitin-mediated proteolysis. This finding suggests that the regulation of ubiquitin-mediated proteolysis of cyclin E is one of the TRIP-Br integrator functions required for proper execution of E2F-dependent cell cycle progression. In addition, prolonged exposure of cells to the decoy peptides induced massive caspase-independent cellular sub-diploidization. The process of decoy peptide-induced sub-diploidization was associated with abnormal stabilization of Geminin, which occurs through a mechanism involving cyclin E deregulation. To study the involvement of the TRIP-Br proteins in the regulation of cellular proliferation, DNA enzymes that specifically suppress the expression of endogenous hTRIP-Br1 (E-Br1) or hTRIP-Br2 (E-Br2), were introduced into quiescent WI-38 human diploid fibroblasts. Both E-Br1 and E-Br2 were capable of efficiently suppressing serum-inducible S phase entry and cellular proliferation in WI-38 cells, consistent with a prior report that the TRIP-Br proteins are required for serum-induced cell cycle progression.